Chemical approaches to study O-GlcNAcylation

Banerjee, P. S.; Hart, Gerald W.; Cho, Jin Won

doi:10.1039/c2cs35412h

Cited by 59 publications

(49 citation statements)

References 65 publications

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“…This moiety is dynamically added and removed by the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) enzymes, respectively (1). UDP-GlcNAc is the donor substrate of OGT, and is biosynthesized through the hexosamine pathway (HBP).…”

Section: Introductionmentioning

confidence: 99%

O-GlcNAcylation is increased in prostate cancer tissues and enhances malignancy of prostate cancer cells

Gao

Han

et al. 2014

Molecular Medicine Reports

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Abstract. O-GlcNAc is an O-linked β-N-acetylglucosamine moiety attached to the side-chain hydroxyl of a serine or threonine residue in numerous cytoplasmic and nuclear proteins. In this study, we detected the level of O-GlcNAc in prostate, liver and pancreatic cancer tissues, and found that the global O-GlcNAc modification also known as O-GlcNAcylation, is specifically increased in prostate cancer tissues compared to corresponding adjacent tissues. In addition, we found that global O-GlcNAcylation is increased in prostate cancer cells and not in benign prostatic hyperplasia (BPH) epithelial cells. O-GlcNAc enhanced the anchorage-independent growth and the migratory/invasive ability of prostate cancer cells. More importantly, we provide here, for the first time to the best of our knowledge, direct evidence that increased O-GlcNAcylation induces malignant transformation of nontumorigenic (BPH) cells. Furthermore, our study suggested that inhibiting the formation of the E-cadherin/catenin/cytoskeleton complex may underly the O-GlcNAc-induced prostate cancer progression. Overall, these findings indicated that O-GlcNAcylation is increased in prostate, but not in liver and pancreatic cancer tissues, and that O-GlcNAc can enhance the malignancy of prostate cancer cells. IntroductionNumerous nuclear and cytoplasmic proteins have been found modified with O-β-N-acetylglucosamine (O-GlcNAc) at the hydroxyl moiety of serine or threonine residues. This moiety is dynamically added and removed by the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) enzymes, respectively (1). UDP-GlcNAc is the donor substrate of OGT, and is biosynthesized through the hexosamine pathway (HBP). The HBP flux is highly dependent on glucose and glutamine, with ~3-5% of total glucose entering this pathway (2). O-GlcNAc is a sensor of intracellular glucose metabolism, since the intracellular level of UDP-GlcNAc is the main regulatory factor for the activity of OGT (3). O-GlcNAcylation is altered in metabolism-associated diseases, such as type II diabetes (4-6), Alzheimer's disease (7) and cancer (8). Therefore, abnormal O-GlcNAcylation may play critical roles in these pathological processes.Aberrant metabolism is a hallmark of cancer. Most cancer cells show increased rates of glucose and glutamine utilization, up to 200-fold higher than those observed in the healthy cells they originate from, and predominantly produce energy through glycolysis followed by lactic acid fermentation (9,10). As a glycolytic pathway, HBP can enhance the level of UDP-GlcNAc, and thus, the activity of OGT, which may lead to an increase in the global O-GlcNAc level (summed over all proteins) in cancer cells. We have previously demonstrated that the global O-GlcNAc level is increased in breast, lung and colon cancer tissues as compared to respective adjacent tissues (11,12). In addition, other research groups have shown that O-GlcNAcylation is increased in breast, prostate and pancreatic cancer cell lines (13-15). However, whether increased O-GlcNAcylation is universal in ...

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Section: Introductionmentioning

confidence: 99%

O-GlcNAcylation is increased in prostate cancer tissues and enhances malignancy of prostate cancer cells

Gao

Han

et al. 2014

Molecular Medicine Reports

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“…Two-hundred and eightyfour GlcNAc-modified chromatin proteins were identified with the use of the RL2 antibody and mass spectrometry based proteomics. Chemical methods have also been designed and successfully applied in the enrichment of O-GlcNAc (19). ␤-elimination of the O-GlcNAc group followed by Michael addition of a dithiothreitol or biotin tag, or named BEMAD, was developed and applied in many studies.…”

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confidence: 99%

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

Wang

Yuan

Fan

et al. 2016

Molecular & Cellular Proteomics

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Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13 C-labeled UDP-GlcNAc from 13 C 6 -glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein OGlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time. Molecular &

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“…One unique subclass of O-glycosylation, found in the cytoplasm and nucleus of most eukaryotic cells, is the addition of a single β -linked GlcNAc residue onto either Ser or Thr (Figure 1c) by the enzyme O-GlcNAc transferase (OGT). 56–58 Termed O-GlcNAcylation, this intracellular form of glycosylation is a rapidly reversible, substoichiometric modification, analogous to phosphorylation. 59 The modification is involved in a number of signaling pathways, including cellular metabolism and physiology.…”

Section: Glycosylation Is An Important Ptm That Influences Proteinmentioning

confidence: 99%

Chemical Glycoproteomics

Palaniappan

Bertozzi²

2016

Chem. Rev.

247

205

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Chemical tools have accelerated progress in glycoscience, reducing experimental barriers to studying protein glycosylation, the most widespread and complex form of posttranslational modification. For example, chemical glycoproteomics technologies have enabled the identification of specific glycosylation sites and glycan structures that modulate protein function in a number of biological processes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. In this review, we describe the important role that chemical glycoproteomics methods are playing in such efforts. We summarize developments in four key areas: enrichment of glycoproteins and glycopeptides from complex mixtures, emphasizing methods that exploit unique chemical properties of glycans or introduce unnatural functional groups through metabolic labeling and chemoenzymatic tagging; identification of sites of protein glycosylation; targeted glycoproteomics; and functional glycoproteomics, with a focus on probing interactions between glycoproteins and glycan-binding proteins. Our goal with this survey is to provide a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.

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Chemical approaches to study O-GlcNAcylation

Cited by 59 publications

References 65 publications

O-GlcNAcylation is increased in prostate cancer tissues and enhances malignancy of prostate cancer cells

O-GlcNAcylation is increased in prostate cancer tissues and enhances malignancy of prostate cancer cells

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

Chemical Glycoproteomics

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